Plerixafor Ex Vivo Mobilization of Placental Derived Haematopoietic Stem Cells

The utility of cord blood as an alternative source of stem cells for haematopoietic stem cell transplantation is well established in the paediatric population. Although it's use in adults has surged over the past few years there are still problems associated with a low stem cell dose which may lead to delayed engraftment, incomplete and inefficient immune reconstitution, and poor survival. Various techniques have been used to increase the stem cell dose including ex vivo expansion, the use of double cords, intrabone injections and the use of third party donor cells. Using standard collection techniques only about 25% of cord blood samples collected reach the target cell dose in adults. The placenta is a rich source of CD34+ cells which may not be fully mobilised using current cord blood collection techniques. A modified two-step collection technique in which the initial collection is followed by placental perfusion with 50 ml heparinised 0.9% saline has shown a 15% increase in the number of nucleated cells. We have modified this technique further by perfusing the placenta with Plerixafor, a CXCR4 antagonist that is currently licensed in the UK for mobilisation of autologous stem cells from adults. The study aim was to assess whether plerixafor could enhance recovery of CD34+ cells and change the immune profile of the perfusate.

Ten cord blood units were collected as per our standard operating procedures. After cord blood collection, 5 placentas were perfused with 50 ml 0.9% saline plus 5,000 units of preservative free heparin by slow infusion over 50 minutes and perfusate collected after about 5 minutes of starting the infusion over 50 minutes (controls). The other 5 placentas were treated the same way except for the addition of 24 mg Plerixafor to the 50 ml 0.9% saline plus 5,000 units of preservative free heparin and collected as per control arm (plerixafor arm).

The samples were analysed by multicolour flow cytometry using a combination of antibody cocktails to identify and enumerate CD34+ stem cells, T cells, T cell precursors, T cell subsets, T regulatory cells, B cells, NK and NK cell precursors, and Dendtritic cells. The CD34+ progenitor cells were further quantified by CFU – a methylcellulose media based assay.

Successful recovery of perfusate was achieved from 9 placentas (mean volume 44 ml +/− SEM 4.7) as one of the collected samples clotted. There was no significant difference in the perfusate volumes between the 2 arms (48ml v 38ml, P=0.152). Percentage increase in numbers of cells recovered by perfusing the placenta was calculated from total numbers of CD34+ and CD45+ cells collected from each cord and placenta pair. Plerixafor did not significantly increase the recovery of cells from the placenta. Perfusion of the 5 control placentas increased recovery of CD34+ cell by 5.06% (mean +/− 0.89 SEM) and CD45+ cells by 4.69% (mean +/−1.09 SEM). Perfusion of 4 placentas with saline plus Plerixafor increased recovery of CD34+ cells by 5.79% (mean +/−2.17 SEM) and CD45+ cells by 2.61% (mean +/− 0.79 SEM). Viability of haematopoietic stem cell progenitors was assessed by colony formation assay with no significant difference detected between the control arm (mean 18.4 CFU +/− 8.26 SEM; n=5) and the plerixafor arm (mean 4.8 CFU +/− 2.7 SEM; n=4).

There was no significant difference in the number of CD34+ cells recovered from the controls as compared to their corresponding cords (n=5, paired t test, p= 0.054). Similarly, there was no significant difference in the plerixafor arm (n=4, paired t test, p=0.091). There was no significant increase in the number of CD34+ stem cells recovered from the plerixafor group as compared to the control group (unpaired t test, p=0.74). There was no significant increase in the number of CD45+ cells in the plerixafor arm compared to the controls (unpaired t test, p= 0.4). Similarly there was no significant difference in the clonal efficiency of the between the 2 groups (unpaired t test, p=0.2).

There were no significant differences in the CD3, CD4, CD8 T cells, Tregs, NK, DCs, progenitor T and NK cells between the 2 groups.

In this pilot study plerixafor does not seem to significantly increase the CD34+ stem cells and other cell subsets. It is possible that the duration of infusion was too short to mobilise the stem cells. Further studies are warranted to investigate this further.

Off Label Use: Plerixafor for stem cell mobilzation from placenta. Shaw:Therakos, a Johnson and Johnson company: Honoraria, Speakers Bureau.